Cloning and Expression Analysis of ClPP2C3 in Citrullus lanatus

doi:10.13560/j.cnki.biotech.bull.1985.2023-0709

Abstract

Abstract:

【Objective】Protein phosphatase (PP2C) is a type of protein phosphatase that exists in both animals and plants, In order to explore its important role in the ripening process of watermelon fruits.【Method】In this study, a PP2C gene ClPP2C3 was cloned in watermelon (Citrullus lanatus) by reverse transcription- polymerase chain reaction (RT-PCR), and its bioinformatics, expression pattern and subcellular localization analysis were conducted. 【Result】The cDNA sequence length of ClPP2C3 in watermelon was 1 317 bp and it encoded 438 amino acids. The molecular weight of ClPP2C3 protein was 47.81 kD and its isoelectric point was 5.12. The ClPP2C3 protein contained one PP2C conserved domain and it had high homology with tomato SlPP2C3 in tomato and FaABI1 in strawberry that negatively regulated fruit ripening. ClPP2C3 in the watermelon was located in the nucleus. The gene quantitative expression analysis showed that the expression of ClPP2C3 in high-sugar-containing watermelons was significantly higher than that in low-sugar-containing watermelons. The 2 kb promoter (up 2 kb to ATG) activity of cultivated watermelon was significantly higher than that in ancestral watermelons, while there was no significant difference in the 1 kb promoter (up 1 kb to ATG) activity detected between these two species. 【Conclusion】The SNPs in 1-2 kb promoter region might result in the difference of promoter activity in different varieties.

Key words: Citrullus lanatus, ClPP2C3, gene cloning, subcellular localization, analysis of promoter activity differences

ZHU Yi, LIU Tang-jing, GONG Guo-yi, ZHANG Jie, WANG Jin-fang, ZHANG Hai-ying. Cloning and Expression Analysis of ClPP2C3 in Citrullus lanatus[J]. Biotechnology Bulletin, 2024, 40(1): 243-249.

Figures/Tables 9

References 16

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功能Function	正向引物序列 Forward primer sequences（5'-3'）	反向引物序列Reverse primer sequences（5'-3'）
克隆基因 Cloning of genes	ATGGCGGAGATTTGCTGTA	TCAAATTAAATTCTTTTGTACTAAATT
荧光定量PCR Quantitative real-time PCR	AGTGGCTAGACGTCGGAGAA	AAATCTCCCGACCGTTTCTT
亚细胞定位 Subcellular localization	TGTGCGATGGATATTTCGCGAATGGCGGAGATTTGCTGTAAA	GATTTCTGGATCCGCAGGCCTAATTAAATTCTTTTGTACTAAATTAAAAA
启动子活性分析 Activity analysis of promotor	0.5k-GTCGACGGTATCGATAAGCTTTAGAAAAAAAACAATTATTTAAATGGGG	TTGTCTAGAACTAGTGGATCCTACTAAGTTCTTTAATCTCCCCTCCC
	1.0k-GTCGACGGTATCGATAAGCTTAGGTTTCATAGAACATTACACATTCAAA
	2.0k-GTCGACGGTATCGATAAGCTTTTTTAATTTTTATTAGCAATTAGCAATTAGA

功能Function	正向引物序列 Forward primer sequences（5'-3'）	反向引物序列Reverse primer sequences（5'-3'）
克隆基因 Cloning of genes	ATGGCGGAGATTTGCTGTA	TCAAATTAAATTCTTTTGTACTAAATT
荧光定量PCR Quantitative real-time PCR	AGTGGCTAGACGTCGGAGAA	AAATCTCCCGACCGTTTCTT
亚细胞定位 Subcellular localization	TGTGCGATGGATATTTCGCGAATGGCGGAGATTTGCTGTAAA	GATTTCTGGATCCGCAGGCCTAATTAAATTCTTTTGTACTAAATTAAAAA
启动子活性分析 Activity analysis of promotor	0.5k-GTCGACGGTATCGATAAGCTTTAGAAAAAAAACAATTATTTAAATGGGG	TTGTCTAGAACTAGTGGATCCTACTAAGTTCTTTAATCTCCCCTCCC
	1.0k-GTCGACGGTATCGATAAGCTTAGGTTTCATAGAACATTACACATTCAAA
	2.0k-GTCGACGGTATCGATAAGCTTTTTTAATTTTTATTAGCAATTAGCAATTAGA

分类 Classification	品种名称 Variety name	含糖量（Brix） Sugar content	基因相对表达量 Relative gene expression
C.lanatus.cultivar	97103	9.4±0.648	1.19±0.098ns
C.lanatus.cultivar	WDM	11.6±1.068	1.28±0.124ns
C.lanatus.cultivar	ZZJM	10.6±0.294	1.24±0.102ns
C.lanatus.cultivar	JMT	11.0±0.355	1.39±0.149*
C.lanatus.cultivar	MG728	11.4±1.471	1.25±0.036ns
C.lanatus.cultivar	JX1M	10.9±0.455	1.36±0.136*
C.mucosospermus	PI595203	2.5±0.432	0.36±0.045****
C.mucosospermus	PI248178	2.8±0.216	0.42±0.029****
C.mucosospermus	PI254740	2.1±0.294	0.32±0.054****
C.colocvynthis	PI220778	1.1±0.216	0.12±0.036****
C.colocvynthis	PI632755	1.7±0.141	0.15±0.022****
C.colocvynthis	PI549161	1.3±0.374	0.13±0.022****
C.colocvynthis	PI296337	1.8±0.432	0.19±0.037****
C.colocvynthis	PI296341-FR	0.7±0.245	0.10±0.029****

分类 Classification	品种名称 Variety name	含糖量（Brix） Sugar content	基因相对表达量 Relative gene expression
C.lanatus.cultivar	97103	9.4±0.648	1.19±0.098ns
C.lanatus.cultivar	WDM	11.6±1.068	1.28±0.124ns
C.lanatus.cultivar	ZZJM	10.6±0.294	1.24±0.102ns
C.lanatus.cultivar	JMT	11.0±0.355	1.39±0.149*
C.lanatus.cultivar	MG728	11.4±1.471	1.25±0.036ns
C.lanatus.cultivar	JX1M	10.9±0.455	1.36±0.136*
C.mucosospermus	PI595203	2.5±0.432	0.36±0.045****
C.mucosospermus	PI248178	2.8±0.216	0.42±0.029****
C.mucosospermus	PI254740	2.1±0.294	0.32±0.054****
C.colocvynthis	PI220778	1.1±0.216	0.12±0.036****
C.colocvynthis	PI632755	1.7±0.141	0.15±0.022****
C.colocvynthis	PI549161	1.3±0.374	0.13±0.022****
C.colocvynthis	PI296337	1.8±0.432	0.19±0.037****
C.colocvynthis	PI296341-FR	0.7±0.245	0.10±0.029****